The invention is directed to novel xcex22 adrenergic receptor agonists. The invention is also directed to pharmaceutical compositions comprising such compounds, methods of using such compounds to treat diseases associated with xcex22 adrenergic receptor activity, and processes and intermediates useful for preparing such compounds.
xcex22 adrenergic receptor agonists are recognized as effective drugs for the treatment of pulmonary diseases such as asthma and chronic obstructive pulmonary disease (including chronic bronchitis and emphysema). xcex22 adrenergic receptor agonists are also useful for treating pre-term labor, and are potentially useful for treating neurological disorders and cardiac disorders. In spite of the success that has been achieved with certain xcex22 adrenergic receptor agonists, current agents possess less than desirable potency, selectivity, onset, and/or duration of action. Thus, there is a need for additional xcex22 adrenergic receptor agonists having improved properties. Preferred agents may possess, among other properties, improved potency, selectivity, onset, and/or duration of action.
The invention provides novel compounds that possess xcex22 adrenergic receptor agonist activity. Accordingly, there is provided a compound of the invention which is a compound of formula (I): 
wherein:
R1 is xe2x80x94CH2OH or xe2x80x94NHCHO, and R2 is hydrogen; or R1 and R2 taken together are xe2x80x94NHC(xe2x95x90O)CHxe2x95x90CHxe2x80x94;
R3, R4, and R5 are each independently hydrogen, halo, alkyl, alkoxy, or aryl, wherein each alkyl and alkoxy is optionally substituted with one or more halo; or R3 and R4 together form a fused benzo ring; or R4 and R5 together form a fused benzo ring; and
R6 is hydrogen or hydroxy;
or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof.
The invention also provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically-acceptable carrier. The invention further provides combinations comprising a compound of the invention and one or more other therapeutic agents and pharmaceutical compositions comprising such combinations.
The invention also provides a method of treating a disease or condition associated with xcex22 adrenergic receptor activity (e.g. a pulmonary disease, such as asthma or chronic obstructive pulmonary disease, pre-term labor, a neurological disorder, a cardiac disorder, or inflammation) in a mammal, comprising administering to the mammal, a therapeutically effective amount of a compound of the invention. The invention further provides a method of treatment comprising administering a therapeutically effective amount of a combination of a compound of the invention together with one or more other therapeutic agents.
The invention also provides a method of treating a disease or condition associated with xcex22 adrenergic receptor activity (e.g. a pulmonary disease, such as asthma or chronic obstructive pulmonary disease, pre-term labor, a neurological disorder, a cardiac disorder, or inflammation) in a mammal, comprising administering to the mammal, a therapeutically effective amount of a pharmaceutical composition of the invention.
This invention also provides a method of modulating a xcex22 adrenergic receptor, the method comprising stimulating a xcex22 adrenergic receptor with a modulatory amount of a compound of the invention.
In separate and distinct aspects, the invention also provides synthetic processes and intermediates described herein, which are useful for preparing compounds of the invention.
The invention also provides a compound of the invention as described herein for use in medical therapy, as well as the use of a compound of the invention in the manufacture of a formulation or medicament for treating a disease or condition associated with xcex22 adrenergic receptor activity (e.g. a pulmonary disease, such as asthma or chronic obstructive pulmonary disease, pre-term labor, a neurological disorder, a cardiac disorder, or inflammation) in a mammal.
When describing the compounds, compositions and methods of the invention, the following terms have the following meanings, unless otherwise indicated.
The term xe2x80x9calkylxe2x80x9d refers to a monovalent saturated hydrocarbon group which may be linear or branched or combinations thereof. Such alkyl groups preferably contain from 1 to 20 carbon atoms; more preferably, from 1 to 8 carbon atoms; and still more preferably, from 1 to 4 carbon atoms. Representative alkyl groups include, by way of example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like.
The term xe2x80x9calkoxyxe2x80x9d refers to a group of the formula xe2x80x94OR, where R is an alkyl group as defined herein. Representative alkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, n-pentoxy, n-hexoxy and the like.
The term xe2x80x9carylxe2x80x9d refers to a monovalent carbocyclic group which may be monocyclic or multicyclic (i.e., fused) wherein at least one ring is aromatic. Such aryl groups preferably contain from 6 to 20 carbon atoms, more preferably, from 6 to 10 carbon atoms. This term includes multicyclic carbocyclic ring systems wherein one or more rings are not aromatic, provided the point of attachment is on an aromatic ring. Representative aryl groups include, by way of example, phenyl, napthyl, azulenyl, indan-5-yl, 1,2,3,4-tetrahydronaphth-6-yl, and the like.
The term xe2x80x9chaloxe2x80x9d refers to a fluoro, chloro, bromo or iodo.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d refers to an amount sufficient to effect treatment when administered to a patient in need of treatment.
The term xe2x80x9ctreatmentxe2x80x9d as used herein refers to the treatment of a disease or medical condition in a patient, such as a mammal (particularly a human) which includes:
(a) preventing the disease or medical condition from occurring, i.e., prophylactic treatment of a patient;
(b) ameliorating the disease or medical condition, i.e., eliminating or causing regression of the disease or medical condition in a patient;
(c) suppressing the disease or medical condition, i.e., slowing or arresting the development of the disease or medical condition in a patient; or
(d) alleviating the symptoms of the disease or medical condition in a patient.
The phrase xe2x80x9cdisease or condition associated with xcex22 adrenergic receptor activityxe2x80x9d includes all disease states and/or conditions that are acknowledged now, or that are found in the future, to be associated with xcex22 adrenergic receptor activity. Such disease states include, but are not limited to, pulmonary diseases, such as asthma and chronic obstructive pulmonary disease (including chronic bronchitis and emphysema), as well as neurological disorders and cardiac disorders. xcex22 adrenergic receptor activity is also known to be associated with pre-term labor (see U.S. Pat. No. 5,872,126) and some types of inflammation (see International Patent Application Publication Number WO 99/30703 and U.S. Pat. No. 5,290,815).
The term xe2x80x9cpharmaceutically-acceptable saltxe2x80x9d refers to a salt prepared from a base or acid which is acceptable for administration to a patient, such as a mammal. Such salts can be derived from pharmaceutically-acceptable inorganic or organic bases and from pharmaceutically-acceptable inorganic or organic acids.
Salts derived from pharmaceutically-acceptable acids include acetic, benzenesulfonic, benzoic, camphosulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic, xinafoic (1-hydroxy-2-naphthoic acid) and the like. Particularly preferred are salts derived from fumaric, hydrobromic, hydrochloric, acetic, sulfuric, methanesulfonic, xinafoic, and tartaric acids.
Salts derived from pharmaceutically-acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc and the like. Particularly preferred are ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically-acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally-occurring amines and the like, such as arginine, betaine, caffeine, choline, N,Nxe2x80x2-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperadine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
The term xe2x80x9csolvatexe2x80x9d refers to a complex or aggregate formed by one or more molecules of a solute, i.e. a compound of the invention or a pharmaceutically-acceptable salt thereof, and one or more molecules of a solvent. Such solvates are typically crystalline solids having a substantially fixed molar ratio of solute and solvent. Representative solvents include by way of example, water, methanol, ethanol, isopropanol, acetic acid, and the like. When the solvent is water, the solvate formed is a hydrate.
It will be appreciated that the term xe2x80x9cor a pharmaceutically-acceptable salt or solvate of stereoisomer thereofxe2x80x9d is intended to include all permutations of salts, solvates and stereoisomers, such as a solvate of a pharmaceutically-acceptable salt of a stereoisomer of a compound of formula (I).
The term xe2x80x9cleaving groupxe2x80x9d refers to a functional group or atom which can be displaced by another functional group or atom in a substitution reaction, such as a nucleophilic substitution reaction. By way of example, representative leaving groups include chloro, bromo and iodo groups; sulfonic ester groups, such as mesylate, tosylate, brosylate, nosylate and the like; and acyloxy groups, such as acetoxy, trifluoroacetoxy and the like.
The term xe2x80x9camino-protecting groupxe2x80x9d refers to a protecting group suitable for preventing undesired reactions at an amino nitrogen. Representative amino-protecting groups include, but are not limited to, formyl; acyl groups, for example alkanoyl groups, such as acetyl; alkoxycarbonyl groups, such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups, such as benzyl (Bn), trityl (Tr), and 1,1-di-(4xe2x80x2-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS); and the like.
The term xe2x80x9chydroxy-protecting groupxe2x80x9d refers to a protecting group suitable for preventing undesired reactions at a hydroxy group. Representative hydroxy-protecting groups include, but are not limited to, alkyl groups, such as methyl, ethyl, and tert-butyl; acyl groups, for example alkanoyl groups, such as acetyl; arylmethyl groups, such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl (benzhydryl, DPM); silyl groups, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS); and the like.
Specific and preferred values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.
A specific value for R3 is hydrogen, alkoxy or halo.
Another specific value for R3 is hydrogen, methoxy, or fluoro.
A specific value for R4 is hydrogen, alkoxy or halo.
Another specific value for R4 is hydrogen, methoxy, or fluoro.
A specific value for R4 and R5 together is a fused benzo ring.
A specific value for R5 is hydrogen, alkyl, aryl, alkoxy or halo.
Another specific value for R5 is hydrogen, fluoro, chloro, methoxy, trifluoromethoxy, difluoromethoxy, 3-methylbutyl, or phenyl.
A preferred group of compounds of formula (I) are compounds wherein R3 is hydrogen or methoxy; R4 is hydrogen or methoxy; and R5 is hydrogen.
Another preferred group of compounds of formula (I) are compounds wherein R3 is hydrogen; R4 is hydrogen; and R5 is hydrogen.
Another preferred group of compounds of formula (I) are compounds wherein R3 is hydrogen; R4 is hydrogen; and R5 is fluoro.
A specific value for R6is hydroxy.
A specific value for R6 is hydrogen.
One preferred compound of formula (I) is a compound of formula (II): 
wherein:
R1 is xe2x80x94CH2OH or xe2x80x94NHCHO, and R2 is hydrogen; or R1 and R2 taken together are xe2x80x94NHC(xe2x95x90O)CHxe2x95x90CHxe2x80x94; and
R3, R4, and R5 are each independently hydrogen, halo, alkyl, alkoxy, or aryl, wherein each alkyl and alkoxy is optionally substituted with one or more halo; or R3 and R4 together form a fused benzo ring; or R4 and R5 together form a fused benzo ring; or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof.
A preferred group of compounds of formula (II) are compounds wherein R3 is hydrogen or methoxy; R4 is hydrogen or methoxy; and R5 is hydrogen.
Another preferred group of compounds of formula (II) are compounds wherein R3 is hydrogen; R4 is hydrogen; and R5 is hydrogen.
Another preferred group of compounds of formula (II) are compounds wherein R3 is hydrogen; R4 is hydrogen; and R5 is fluoro.
Another preferred compound of formula (I) is a compound of formula (III): 
wherein: R3, R4, and R5 are each independently hydrogen, halo, alkyl, alkoxy, or aryl, wherein each alkyl and alkoxy is optionally substituted with one or more halo; or R3 and R4 together form a fused benzo ring; or R4 and R5 together form a fused benzo ring; and R6 is hydrogen or hydroxy; or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof.
Another preferred compound of formula (I) is a compound of formula (IV): 
wherein: R3, R4, and R5 are each independently hydrogen, halo, alkyl, alkoxy, or aryl, wherein each alkyl and alkoxy is optionally substituted with one or more halo; or R3 and R4 together form a fused benzo ring; or R4 and R5 together form a fused benzo ring; and R6 is hydrogen or hydroxy; or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof.
Another preferred compound of formula (I) is a compound of formula (V): 
wherein: R3, R4, and R5 are each independently hydrogen, halo, alkyl, alkoxy, or aryl, wherein each alkyl and alkoxy is optionally substituted with one or more halo; or R3 and R4 together form a fused benzo ring; or R4 and R5 together form a fused benzo ring; and R6 is hydrogen or hydroxy; or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof.
Exemplary compounds of the invention are compounds 1-25 as described in the Examples below. Preferred compounds of the invention are compounds 1-6, 8-11, 14-16, and 18-25. A more preferred sub-group of compounds is compounds 1, 2, 4-6, 19, 20, 22-25.
Particular mention may be made of the following compounds for which the compound numbers are indicated in parentheses:
4-((R)-1-Hydroxy-2-{2-[4-((R)-2-hydroxy-2-phenyl-ethoxy)-phenyl]-ethylamino}-ethyl)-2-hydroxymethyl-phenol (20);
4-[(R)-1-Hydroxy-2-(2-{4-[(R)-2-hydroxy-2-(2-methoxy-phenyl)-ethoxy]-phenyl}-ethylamino)-ethyl]-2-hydroxymethyl-phenol (21);
N-[2-Hydroxy-5-((R)-1-hydroxy-2-{2-[4-((R)-2-hydroxy-2-phenyl-ethoxy)-phenyl]-ethylamino}-ethyl)-phenyl]-formamide (22);
N-{2-Hydroxy-5-[(R)-1-hydroxy-2-(2-{4-[(R)-2-hydroxy-2-(2-methoxy-phenyl)-ethoxy]-ethylamino}ehtyl]-phenyl}-formamide (23);
8-Hydroxy-5-((R)-1-hydroxy-2-{2-[4-((R)-2-hydroxy-2-phenyl-ethoxy)-phenyl]-ethylamino}-ethyl)-1H-quinolin-2-one (24); and
8-Hydroxy-5-[(R)-1-hydroxy-2-(2-{4-[(R)-2-hydroxy-2-(2-methoxy-phenyl)-ethoxy]-phenyl}-ethylamino)-ethyl]-1H-quinolin-2-one (25). (25).
The compounds of the invention may contain a chiral center. Accordingly, the invention includes racemic mixtures, enantiomers, and mixtures enriched in one or more stereoisomer. The scope of the invention as described and claimed encompasses the racemic forms of the compounds as well as the individual enantiomers, diastereomers, and stereoisomer-enriched mixtures. A preferred compound of the invention is a compound that is a mixture of enantiomers, wherein the amount of the (R) enantiomer at the chiral center in formula (I) or formula (II) that is attached to the phenyl ring substituted with R1 and R2 is greater than the amount of the (S) enantiomer. A more preferred compound of the invention is a compound that is the (R) enantiomer at this center.
When R6 is hydroxy, or, equivalently, when the compound is a compound of formula (II), another preferred compound of the invention is a compound that is a mixture of enantiomers, wherein the amount of the (R) enantiomer at the chiral center attached to the phenyl ring substituted with R3, R4, and R5 (i.e. in formula (I), the chiral center substituted with R6) is greater than the amount of the (S) enantiomer at this center. A more preferred compound of the invention is a compound that is the (R) enantiomer at this center.
Further, when R6 is hydroxy, or the compound is a compound of formula (II), a preferred diastereomer of the invention is a compound that is a mixture of diastereomers, wherein the amount of the (R, R) diastereomer at the two chiral centers identified above in formula (I) or (II) is greater than the amount of other diastereomers. A more preferred compound of the invention when R6 is hydroxy, or when the compound is a compound of formula (II), is a compound that is the (R, R) diastereomer at the two chiral centers of formula (I) or formula (II).
General Synthetic Procedures
The compounds of the invention can be prepared using the methods and procedures described herein, or using similar methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group, as well as suitable conditions for protection and deprotection, are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.
Processes for preparing compounds of the invention are provided as further embodiments of the invention and are illustrated by the procedures below.
For example, a compound of formula (I) can be prepared from a corresponding intermediate compound of formula (VI): 
wherein Ra is a amino-protecting group (e.g. benzyl) and Rb is hydrogen, by removing the protecting group Ra to provide the compound of formula (I).
A compound of formula (I) can also be prepared from a corresponding intermediate compound of formula (VI) wherein Ra is hydrogen and Rb is a hydroxy-protecting group (e.g. benzyl), by removing the protecting group Rb to provide the compound of formula (I).
A compound of formula (I) can also be prepared from a corresponding intermediate compound of formula (VI) wherein Ra is a amino-protecting group (e.g. benzyl) and Rb is a hydroxy-protecting group (e.g. benzyl), by removing the protecting groups (Ra and Rb) to provide the compound of formula (I).
An intermediate compound of formula (VI) can be prepared by alkylating a corresponding alcohol of formula (VII) with a corresponding compound of formula (VIII), wherein Z is a suitable leaving group (e.g. bromo, iodo, tosyl, or mesyl). 
Suitable conditions for such alkylations are well known, and are illustrated in the Examples hereinbelow.
Accordingly, the invention provides a method for preparing a compound of formula (VI): 
wherein Ra is a amino-protecting group (e.g. benzyl); Rb is a hydroxy-protecting group (e.g. benzyl); and R1-R6 have any of the values, specific values or preferred values described herein, comprising alkylating a corresponding alcohol of formula (VII) with a corresponding compound of formula (VIII): 
wherein Z is a suitable leaving group (e.g. bromo, iodo, tosyl, or mesyl), to provide the compound of formula (VI).
The invention also provides novel compounds of formulae (VI-VIII) described herein.
Pharmaceutical Compositions
The invention also provides pharmaceutical compositions comprising a compound of the invention. Accordingly, the compound, preferably in the form of a pharmaceutically-acceptable salt, can be formulated for any suitable form of administration, such as oral or parenteral administration, or administration by inhalation.
By way of illustration, the compound can be admixed with conventional pharmaceutical carriers and excipients and used in the form of powders, tablets, capsules, elixirs, suspensions, syrups, wafers, and the like. Such pharmaceutical compositions will contain from about 0.05 to about 90% by weight of the active compound, and more generally from about 0.1 to about 30%. The pharmaceutical compositions may contain common carriers and excipients, such as cornstarch or gelatin, lactose, magnesium sulfate, magnesium stearate, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and alginic acid. Disintegrators commonly used in the formulations of this invention include croscarmellose, microcrystalline cellulose, cornstarch, sodium starch glycolate and alginic acid.
A liquid composition will generally consist of a suspension or solution of the compound or pharmaceutically-acceptable salt in a suitable liquid carrier(s), for example ethanol, glycerine, sorbitol, non-aqueous solvent such as polyethylene glycol, oils or water, optionally with a suspending agent, a solubilizing agent (such as a cyclodextrin), preservative, surfactant, wetting agent, flavoring or coloring agent. Alternatively, a liquid formulation can be prepared from a reconstitutable powder.
For example a powder containing active compound, suspending agent, sucrose and a sweetener can be reconstituted with water to form a suspension; a syrup can be prepared from a powder containing active ingredient, sucrose and a sweetener.
A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid compositions. Examples of such carriers include magnesium stearate, starch, lactose, sucrose, microcrystalline cellulose and binders, for example polyvinylpyrrolidone. The tablet can also be provided with a color film coating, or color included as part of the carrier(s). In addition, active compound can be formulated in a controlled release dosage form as a tablet comprising a hydrophilic or hydrophobic matrix.
A composition in the form of a capsule can be prepared using routine encapsulation procedures, for example by incorporation of active compound and excipients into a hard gelatin capsule. Alternatively, a semi-solid matrix of active compound and high molecular weight polyethylene glycol can be prepared and filled into a hard gelatin capsule; or a solution of active compound in polyethylene glycol or a suspension in edible oil, for example liquid paraffin or fractionated coconut oil can be prepared and filled into a soft gelatin capsule.
Tablet binders that can be included are acacia, methylcellulose, sodium carboxymethylcellulose, poly-vinylpyrrolidone (Povidone), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose. Lubricants that can be used include magnesium stearate or other metallic stearates, stearic acid, silicone fluid, talc, waxes, oils and colloidal silica.
Flavoring agents such as peppermint, oil of wintergreen, cherry flavoring or the like can also be used. Additionally, it may be desirable to add a coloring agent to make the dosage form more attractive in appearance or to help identify the product.
The compounds of the invention and their pharmaceutically-acceptable salts that are active when given parenterally can be formulated for intramuscular, intrathecal, or intravenous administration.
A typical composition for intra-muscular or intrathecal administration will consist of a suspension or solution of active ingredient in an oil, for example arachis oil or sesame oil. A typical composition for intravenous or intrathecal administration will consist of a sterile isotonic aqueous solution containing, for example active ingredient and dextrose or sodium chloride, or a mixture of dextrose and sodium chloride. Other examples are lactated Ringer""s injection, lactated Ringer""s plus dextrose injection, Normosol-M and dextrose, Isolyte E, acylated Ringer""s injection, and the like. Optionally, a co-solvent, for example, polyethylene glycol; a chelating agent, for example, ethylenediamine tetraacetic acid; a solubilizing agent, for example, a cyclodextrin; and an anti-oxidant, for example, sodium metabisulphite, may be included in the formulation. Alternatively, the solution can be freeze dried and then reconstituted with a suitable solvent just prior to administration.
The compounds of this invention and their pharmaceutically-acceptable salts which are active on topical administration can be formulated as transdermal compositions or transdermal delivery devices (xe2x80x9cpatchesxe2x80x9d). Such compositions include, for example, a backing, active compound reservoir, a control membrane, liner and contact adhesive. Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, for example, U.S. Pat. No. 5,023,252. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
One preferred manner for administering a compound of the invention is inhalation. Inhalation is an effective means for delivering an agent directly to the respiratory tract. There are three general types of pharmaceutical inhalation devices: nebulizer inhalers, dry powder inhalers (DPI), and metered-dose inhalers (MDI). Nebulizer devices produce a stream of high velocity air that causes a therapeutic agent to spray as a mist which is carried into the patient""s respiratory tract. The therapeutic agent is formulated in a liquid form such as a solution or a suspension of micronized particles of respirable size, where micronized is typically defined as having about 90% or more of the particles with a diameter of less than about 10 xcexcm. A typical formulation for use in a conventional nebulizer device is an isotonic aqueous solution of a pharmaceutical salt of the active agent at a concentration of the active agent of between about 0.05 xcexcg/mL and about 10 mg/mL.
DPI""s typically administer a therapeutic agent in the form of a free flowing powder that can be dispersed in a patient""s air-stream during inspiration. In order to achieve a free flowing powder, the therapeutic agent can be formulated with a suitable excipient, such as lactose or starch. A dry powder formulation can be made, for example, by combining dry lactose having a particle size between about 1 xcexcm and about 100 xcexcm with micronized particles of a pharmaceutical salt of the active agent and dry blending. Alternative, the agent can be formulated without excipients. The formulation is loaded into a dry powder dispenser, or into inhalation cartridges or capsules for use with a dry powder delivery device.
Examples of DPI delivery devices provided commercially include Diskhaler (GlaxoSmithKline, Research Triangle Park, N.C.) (see, e.g., U.S. Pat. No. 5,035,237); Diskus (GlaxoSmithKline) (see, e.g., U.S. Pat. No. 6,378,519; Turbuhaler (AstraZeneca, Wilmington, Del.) (see, e.g., U.S. Pat. No. 4,524,769); and Rotahaler (GlaxoSmithKline) (see, e.g., U.S. Pat. No. 4,353,365). Further examples of suitable DPI devices are described in U.S. Pat. Nos. 5,415,162, 5,239,993, and 5,715,810 and references therein.
MDI""s typically discharge a measured amount of therapeutic agent using compressed propellant gas. Formulations for MDI administration include a solution or suspension of active ingredient in a liquefied propellant. While chlorofluorocarbons, such as CCl3F, conventionally have been used as propellants, due to concerns regarding adverse affects of such agents on the ozone layer, formulations using hydrofluoroalklanes (HFA), such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3,-heptafluoro-n-propane, (HFA 227) have been developed. Additional components of HFA formulations for MDI administration include co-solvents, such as ethanol or pentane, and surfactants, such as sorbitan trioleate, oleic acid, lecithin, and glycerin. (See, for example, U.S. Pat. No. 5,225,183, EP 0717987 A2, and WO 92/22286.)
Thus, a suitable formulation for MDI administration can include from about 0.01% to about 5% by weight of a pharmaceutical salt of active ingredient, from about 0% to about 20% by weight ethanol, and from about 0% to about 5% by weight surfactant, with the remainder being the HFA propellant. In one approach, to prepare the formulation, chilled or pressurized hydrofluoroalkane is added to a vial containing the pharmaceutical salt of active compound, ethanol (if present) and the surfactant (if present). To prepare a suspension, the pharmaceutical salt is provided as micronized particles. The formulation is loaded into an aerosol canister, which forms a portion of an MDI device. Examples of MDI devices developed specifically for use with HFA propellants are provided in U.S. Pat. Nos. 6,006,745 and 6,143,277.
In an alternative preparation, a suspension formulation is prepared by spray drying a coating of surfactant on micronized particles of a pharmaceutical salt of active compound. (See, for example, WO 99/53901 and WO 00/61108.) For additional examples of processes of preparing respirable particles, and formulations and devices suitable for inhalation dosing see U.S. Pat. Nos. 6,268,533, 5,983,956, 5,874,063, and 6,221,398, and WO 99/55319 and WO 00/30614.
It will be understood that any form of the compounds of the invention, (i.e. free base, pharmaceutical salt, or solvate) that is suitable for the particular mode of administration, can be used in the pharmaceutical compositions discussed above.
The active compounds are effective over a wide dosage range and are generally administered in a therapeutically effective amount. It will be understood, however, that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient""s symptoms, and the like.
Suitable doses of the therapeutic agents for inhalation administration are in the general range of from about 0.05 xcexcg/day to about 1000 xcexcg/day, preferably from about 0.5 xcexcg/day to about 500 xcexcg/day. A compound can be administered in a periodic dose: weekly, multiple times per week, daily, or multiple doses per day. The treatment regimen may require administration over extended periods of time, for example, for several weeks or months, or the treatment regimen may require chronic administration. Suitable doses for oral administration are in the general range of from about 0.05 xcexcg/day to about 100 mg/day, preferably 0.5 to 1000 xcexcg/day.
The present active agents can also be co-administered with one or more other therapeutic agents. For example, the present agents can be administered in combination with one or more therapeutic agents selected from anti-inflammatory agents (e.g. corticosteroids and non-steroidal anti-inflammatory agents (NSAIDs), antichlolinergic agents (particularly muscarinic receptor antagonists), other xcex22 adrenergic receptor agonists, antiinfective agents (e.g. antibiotics or antivirals) or antihistamines. The invention thus provides, in a further aspect, a combination comprising a compound of the invention together with one or more therapeutic agent, for example, an anti-inflammatory agent, an antichlolinergic agent, another xcex22 adrenergic receptor agonist, an antiinfective agent or an antihistamine.
The other therapeutic agents can be used in the form of pharmaceutically-acceptable salts or solvates. As appropriate, the other therapeutic agents can be used as optically pure stereoisomers.
Suitable anti-inflammatory agents include corticosteroids and NSAIDs. Suitable corticosteroids which may be used in combination with the compounds of the invention are those oral and inhaled corticosteroids and their pro-drugs which have anti-inflammatory activity. Examples include methyl prednisolone, prednisolone, dexamethasone, fluticasone propionate, 6xcex1,9xcex1-difluoro-17xcex1-[(2-furanylcarbonyl)oxy]-11xcex2-hydroxy-16xcex1-methyl-3-oxo-androsta-1,4-diene-17xcex2-carbothioic acid S-fluoromethyl ester, 6xcex1,9xcex1-difluoro-11xcex2-hydroxy-16xcex1-methyl-3-oxo-17xcex1-propionyloxy-androsta-1,4-diene-17xcex2-carbothioic acid S-(2-oxo-tetrahydro-furan-3S-yl) ester, beclomethasone esters (e.g. the 17-propionate ester or the 17,21-dipropionate ester), budesonide, flunisolide, mometasone esters (e.g. the furoate ester), triamcinolone acetonide, rofleponide, ciclesonide, butixocort propionate, RPR-106541, and ST-126. Preferred corticosteroids include fluticasone propionate, 6xcex1,9xcex1-difluoro-11xcex2-hydroxy-16xcex1-methyl-17xcex1-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-17xcex2-carbothioic acid S-fluoromethyl ester and 6xcex1,9xcex1-difluoro-17xcex1-[(2-furanylcarbonyl)oxy]-11xcex2-hydroxy-16xcex1-methyl-3-oxo-androsta-1,4-diene-17xcex2-carbothioic acid S-fluoromethyl ester, more preferably 6xcex1,9xcex1-difluoro-17xcex1-[(2-furanylcarbonyl)oxy]-11xcex2-hydroxy-16xcex1-methyl-3-oxo-androsta-1,4-diene-17xcex2-carbothioic acid acid S-fluoromethyl ester.
Suitable NSAIDs include sodium cromoglycate; nedocromil sodium; phosphodiesterase (PDE) inhibitors (e.g. theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors); leukotriene antagonists (e.g. monteleukast); inhibitors of leukotriene synthesis; iNOS inhibitors; protease inhibitors, such as tryptase and elastase inhibitors; beta-2 integrin antagonists and adenosine receptor agonists or antagonists (e.g. adenosine 2a agonists); cytokine antagonists (e.g. chemokine antagonists such as, an interleukin antibody (xcex1IL antibody), specifically, an xcex1IL-4 therapy, an xcex1IL-13 therapy, or a combination thereof); or inhibitors of cytokine synthesis. Suitable other xcex22-adrenoreceptor agonists include salmeterol (e.g. as the xinafoate), salbutamol (e.g. as the sulphate or the free base), formoterol (e.g. as the fumarate), fenoterol or terbutaline and salts thereof.
Also of interest is use of the present active agent in combination with a phosphodiesterase 4 (PDE4) inhibitor or a mixed PDE3/PDE4 inhibitor. The PDE4-specific inhibitor useful in this aspect of the invention may be any compound that is known to inhibit the PDE4 enzyme or which is discovered to act as a PDE4 inhibitor, and which are only PDE4 inhibitors. Preferred compounds are cis 4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylic acid, 2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one and cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol].
Other compounds of interest include:
Compounds set out in U.S. Pat. No. 5,552,438 issued 03 Sep. 1996; this patent and the compounds it discloses are incorporated herein in full by reference. The compound of particular interest, which is disclosed in U.S. Pat. No. 5,552,438, is cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylic acid (also known as cilomalast) and its salts, esters, pro-drugs or physical forms;
AWD-12-281 from elbion (Hofgen, N. et al. 15th EFMC Int Symp Med Chem (September 6-10, Edinburgh) 1998, Abst P.98; CAS reference No. 247584020-9); a 9-benzyladenine derivative nominated NCS-613 (INSERM); D-4418 from Chiroscience and Schering-Plough; a benzodiazepine PDE4 inhibitor identified as CI-1018 (PD-168787) and attributed to Pfizer; a benzodioxole derivative disclosed by Kyowa Hakko in WO99/16766; K-34 from Kyowa Hakko; V-11294A from Napp (Landells, L. J. et al. Eur Resp J [Annu Cong Eur Resp Soc (September 19-23, Geneva) 1998] 1998, 12 (Suppl. 28): Abst P2393); roflumilast (CAS reference No 162401-32-3) and a pthalazinone (WO99/47505, the disclosure of which is hereby incorporated by reference) from Byk-Gulden; Pumafentrine, (xe2x88x92)-p-[(4aR*,10bS*)-9-ethoxy-1,2,3,4,4a,10b-hexahydro-8-methoxy-2-methylbenzo[c][1,6]naphthyridin-6-yl]-N,N-diisopropylbenzamide which is a mixed PDE3/PDE4 inhibitor which has been prepared and published on by Byk-Gulden, now Altana; arofylline under development by Almirall-Prodesfarma; VM554/UM565 from Vernalis; or T-440 (Tanabe Seiyaku; Fuji, K. et al. J Pharmacol Exp Ther,1998, 284(1): 162), and T2585.
Other possible PDE-4 and mixed PDE3/PDE4 inhibitors include those listed in WO01/13953, the disclosure of which is hereby incorporated by reference.
Suitable anticholinergic agents are those compounds that act as antagonists at the muscarinic receptor, in particular those compounds which are antagonists of the M1, M2, or M3 receptors, or of combinations thereof. Exemplary compounds include the alkaloids of the belladonna plants as illustrated by the likes of atropine, scopolamine, homatropine, hyoscyamine; these compounds are normally administered as a salt, being tertiary amines. These drugs, particularly the salt forms, are readily available from a number of commercial sources or can be made or prepared from literature data via, to wit:
Atropinexe2x80x94CAS-51-55-8 or CAS-51-48-1 (anhydrous form), atropine sulfatexe2x80x94CAS-5908-99-6; atropine oxidexe2x80x94CAS-4438-22-6 or its HCl saltxe2x80x94CAS-4574-60-1 and methylatropine nitratexe2x80x94CAS-52-88-0.
Homatropinexe2x80x94CAS-87-00-3, hydrobromide saltxe2x80x94CAS-51-56-9, methylbromide saltxe2x80x94CAS-80-49-9.
Hyoscyamine (d, l)xe2x80x94CAS-101-31-5, hydrobromide saltxe2x80x94CAS-306-03-6 and sulfate saltxe2x80x94CAS-6835-16-1.
Scopolaminexe2x80x94CAS-51-34-3, hydrobromide saltxe2x80x94CAS-6533-68-2, methylbromide saltxe2x80x94CAS-155-41-9.
Preferred anticholinergics include ipratropium (e.g. as the bromide), sold under the name Atrovent, oxitropium (e.g. as the bromide) and tiotropium (e.g. as the bromide) (CAS-139404-48-1). Also of interest are: methantheline (CAS-53-46-3), propantheline bromide (CAS-50-34-9), anisotropine methyl bromide or Valpin 50 (CAS-80-50-2), clidinium bromide (Quarzan, CAS-3485-62-9), copyrrolate (Robinul), isopropamide iodide (CAS-71-81-8), mepenzolate bromide (U.S. Pat. No. 2,918,408), tridihexethyl chloride (Pathilone, CAS-4310-35-4), and hexocyclium methylsulfate (Tral, CAS-115-63-9). See also cyclopentolate hydrochloride (CAS-5870-29-1), tropicamide (CAS-1508-75-4), trihexyphenidyl hydrochloride (CAS-144-11-6), pirenzepine (CAS-29868-97-1), telenzepine (CAS-80880-90-9), AF-DX 116, or methoctramine, and the compounds disclosed in WO01/04118, the disclosure of which is hereby incorporated by reference.
Suitable antihistamines (also referred to as H1-receptor antagonists) include any one or more of the numerous antagonists known which inhibit H1-receptors, and are safe for human use. All are reversible, competitive inhibitors of the interaction of histamine with H1-receptors. The majority of these inhibitors, mostly first generation antagonists, are characterized, based on their core structures, as ethanolamines, ethylenediamines, and alkylamines. In addition, other first generation antihistamines include those which can be characterized as based on piperizine and phenothiazines. Second generation antagonists, which are non-sedating, have a similar structure-activity relationship in that they retain the core ethylene group (the alkylamines) or mimic a tertiary amine group with piperizine or piperidine. Exemplary antagonists are as follows:
Ethanolamines: carbinoxamine maleate, clemastine fumarate, diphenylhydramine hydrochloride, and dimenhydrinate.
Ethylenediamines: pyrilamine amleate, tripelennamine HCl, and tripelennamine citrate.
Alkylamines: chlorpheniramine and its salts such as the maleate salt, and acrivastine.
Piperazines: hydroxyzine HCl, hydroxyzine pamoate, cyclizine HCl, cyclizine lactate, meclizine HCl, and cetirizine HCl.
Piperidines: Astemizole, levocabastine HCl, loratadine or its descarboethoxy analogue, and terfenadine and fexofenadine hydrochloride or another pharmaceutically-acceptable salt.
Azelastine hydrochloride is yet another H1 receptor antagonist which may be used in combination with a compound of the invention.
Examples of preferred anti-histamines include methapyrilene and loratadine.
The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof and a corticosteroid. In particular, the invention provides a combination wherein the corticosteroid is fluticasone propionate or wherein the corticosteroid is 6xcex1,9xcex1-difluoro-17xcex1-[(2-furanylcarbonyl)oxy]-11xcex2-hydroxy-16xcex1-methyl-3-oxo-androsta-1,4-diene-17xcex2-carbothioic acid S-fluoromethyl ester or 6xcex1,9xcex1-difluoro-11xcex2-hydroxy-16xcex1-methyl-3-oxo-17xcex1-propionyloxy-androsta-1,4-diene-17xcex2-carbothioic acid S-(2-oxo-tetrahydro-furan-3S-yl) ester.
The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof and a PDE4 inhibitor.
The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof and an anticholinergic agent.
The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof and an antihistamine.
The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof together with a PDE4 inhibitor and a corticosteroid.
The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof together with an anticholinergic agent and a corticosteroid.
As used in the above combinations, the term, xe2x80x9ca compound of formula (I)xe2x80x9d includes a compound of formulas (II), (III), (IV), or (V), and preferred groups thereof, and any individually disclosed compound or compounds.
Accordingly, the pharmaceutical compositions of the invention can optionally comprise combinations of a compound of formula (I) or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof with one or more other therapeutic agents, as described above.
The individual compounds of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. Appropriate doses of known therapeutic agents will be readily appreciated by those skilled in the art. Methods of treatment of the invention, therefore, include administration of the individual compounds of such combinations either sequentially or simultaneously in separate or combined pharmaceutical formulations.
Thus, according to a further aspect, the invention provides a method of treating a disease or condition associated with xcex22 adrenergic receptor activity in a mammal, comprising administering to the mammal a therapeutically effective amount of a combination of a compound of formula (I) or a pharmaceutically-acceptable salt or solvate or stereoisomer thereof with one or more other therapeutic agents.
Additional suitable carriers for formulations of the active compounds of the present invention can be found in Remington: The Science and Practice of Pharmacy, 20th Edition, Lippincott Williams and Wilkins, Philadelphia, Pa., 2000. The following non-limiting examples illustrate representative pharmaceutical compositions of the invention.
This example illustrates the preparation of a representative pharmaceutical composition for oral administration of a compound of this invention:
The above ingredients are mixed and introduced into a hard-shell gelatin capsule.
This example illustrates the preparation of another representative pharmaceutical composition for oral administration of a compound of this invention:
The above ingredients are mixed intimately and pressed into single scored tablets.
This example illustrates the preparation of a representative pharmaceutical composition for oral administration of a compound of this invention.
An oral suspension is prepared having the following composition.
This example illustrates the preparation of a representative pharmaceutical composition containing a compound of this invention.
An injectable preparation buffered to a pH of 4 is prepared having the following composition:
This example illustrates the preparation of a representative pharmaceutical composition for injection of a compound of this invention.
A reconstituted solution is prepared by adding 20 mL of sterile water to 1 mg of the compound of this invention. Before use, the solution is then diluted with 200 mL of an intravenous fluid that is compatible with the active compound. Such fluids are chosen from 5% dextrose solution, 0.9% sodium chloride, or a mixture of 5% dextrose and 0.9% sodium chloride. Other examples are lactated Ringer""s injection, lactated Ringer""s plus 5% dextrose injection, Normosol-M and 5% dextrose, Isolyte E, and acylated Ringer""s injection.
This example illustrates the preparation of a representative pharmaceutical composition containing a compound of this invention.
An injectable preparation is prepared having the following composition:
The above ingredients are blended and the pH is adjusted to 3.5xc2x10.5 using 0.5 N HCl or 0.5 N NaOH.
This example illustrates the preparation of a representative pharmaceutical composition for topical application of a compound of this invention.
All of the above ingredients, except water, are combined and heated to 60xc2x0 C. with stirring. A sufficient quantity of water at 60xc2x0 C. is then added with vigorous stirring to emulsify the ingredients, and water then added q.s. 100 g.
This example illustrates the preparation of a representative pharmaceutical composition containing a compound of the invention.
An aqueous aerosol formulation for use in a nebulizer is prepared by dissolving 0.1 mg of a pharmaceutical salt of active compound in a 0.9% sodium chloride solution acidified with citric acid. The mixture is stirred and sonicated until the active salt is dissolved. The pH of the solution is adjusted to a value in the range of from 3 to 8 by the slow addition of NaOH.
This example illustrates the preparation of a dry powder formulation containing a compound of the invention for use in inhalation cartridges.
Gelatin inhalation cartridges are filled with a pharmaceutical composition having the following ingredients:
The pharmaceutical salt of active compound is micronized prior to blending with lactose. The contents of the cartridges are administered using a powder inhaler.
This example illustrates the preparation of a dry powder formulation containing a compound of the invention for use in a dry powder inhalation device.
A pharmaceutical composition is prepared having a bulk formulation ratio of micronized pharmaceutical salt to lactose of 1:200. The composition is packed into a dry powder inhalation device capable of delivering between about 10 xcexcg and about 100 xcexcg of active drug ingredient per dose.
This example illustrates the preparation of a formulation containing a compound of the invention for use in a metered dose inhaler.
A suspension containing 5% pharmaceutical salt of active compound, 0.5% lecithin, and 0.5% trehalose is prepared by dispersing 5 g of active compound as micronized particles with mean size less than 10 xcexcm in a colloidal solution formed from 0.5 g of trehalose and 0.5 g of lecithin dissolved in 100 mL of demineralized water. The suspension is spray dried and the resulting material is micronized to particles having a mean diameter less than 1.5 xcexcm. The particles are loaded into canisters with pressurized 1,1,1,2-tetrafluoroethane.
This example illustrates the preparation of a formulation containing a compound of the invention for use in a metered dose inhaler.
A suspension containing 5% pharmaceutical salt of active compound and 0.1% lecithin is prepared by dispersing 10 g of active compound as micronized particles with mean size less than 10 xcexcm in a solution formed from 0.2 g of lecithin dissolved in 200 mL of demineralized water. The suspension is spray dried and the resulting material is micronized to particles having a mean diameter less than 1.5 xcexcm. The particles are loaded into canisters with pressurized 1,1,1,2,3,3,3-heptafluoro-n-propane.
Biological Assays
The compounds of this invention, and their pharmaceutically-acceptable salts, exhibit biological activity and are useful for medical treatment. The ability of a compound to bind to the xcex22 adrenergic receptor, as well as its selectivity, agonist potency, and intrinsic activity can be demonstrated using Tests A-B below, or can be demonstrated using other tests that are known in the art.
HEK-293 derived cell lines stably expressing cloned human xcex21 or xcex22 adrenergic receptors, respectively, were grown to near confluency in DMEM with 10% dialyzed FBS in the presence of 500 xcexcg/mL Geneticin. The cell monolayer was lifted with Versene 1:5,000 (0.2 g/L EDTA in PBS) using a cell scraper. Cells were pelleted by centrifugation at 1,000 rpm, and cell pellets were either stored frozen at xe2x88x9280xc2x0 C. or membranes were prepared immediately. For preparation, cell pellets were resuspended in lysis buffer (10 mM Tris/HCL pH 7.4 @ 4xc2x0 C., one tablet of xe2x80x9cComplete Protease Inhibitor Cocktail Tablets with 2 mM EDTAxe2x80x9d per 50 mL buffer (Roche cat. #1697498, Roche Molecular Biochemicals, Indianapolis, Ind.)) and homogenized using a tight-fitting Dounce glass homogenizer (20 strokes) on ice. The homogenate was centrifuged at 20,000xc3x97g, the pellet was washed once with lysis buffer by resuspension and centrifugation as above. The final pellet was resuspended in membrane buffer (75 mM Tris/HCl pH 7.4, 12.5 mM MgCl2, 1 mM EDTA @ 25xc2x0 C.). Protein concentration of the membrane suspension was determined by the method of Bradford (Bradford M M., Analytical Biochemistry, 1976, 72, 248-54). Membranes were stored frozen in aliquots at xe2x88x9280xc2x0 C.
Binding assays were performed in 96-well microtiter plates in a total assay volume of 100 xcexcL with 5 xcexcg membrane protein for membranes containing the human xcex22 adrenergic receptor, or 2.5 xcexcg membrane protein for membranes containing the human xcex21 adrenergic receptor in assay buffer (75 mM Tris/HCl pH 7.4 @ 25xc2x0 C., 12.5 mM MgCl2, 1 mM EDTA, 0.2% BSA). Saturation binding studies for determination of Kd values of the radioligand were done using [3H]dihydroalprenolol (NET-720, 100 Ci/mmol, PerkinElmer Life Sciences Inc., Boston, Mass.) at 10 different concentrations ranging from 0.01 nM-200 nM. Displacement assays for determination of pKi values of compounds were done with [3H]dihydroalprenolol at 1 nM and 10 different concentrations of compound ranging from 40 pM-10 xcexcM. Compounds were dissolved to a concentration of 10 mM in dissolving buffer (25 mM Gly-HCl pH 3.0 with 50% DMSO), then diluted to 1 mM in 50 mM Gly-HCl pH 3.0, and from there serially diluted into assay buffer. Non-specific binding was determined in the presence of 10 xcexcM unlabeled alprenolol. Assays were incubated for 90 minutes at room temperature, binding reactions were terminated by rapid filtration over GF/B glass fiber filter plates (Packard BioScience Co., Meriden, Conn.) presoaked in 0.3% polyethyleneimine. Filter plates were washed three times with filtration buffer (75 mM Tris/HCl pH 7.4 @ 4xc2x0 C., 12.5 mM MgCl2, 1 mM EDTA) to remove unbound radioactivity. Plates were dried, 50 xcexcL Microscint-20 liquid scintillation fluid (Packard BioScience Co., Meriden, Conn.) was added and plates were counted in a Packard Topcount liquid scintillation counter (Packard BioScience Co., Meriden, Conn.). Binding data were analyzed by nonlinear regression analysis with the GraphPad Prism Software package (GraphPad Software, Inc., San Diego, Calif.) using the 3-parameter model for one-site competition. The curve minimum was fixed to the value for nonspecific binding, as determined in the presence of 10 xcexcM alprenolol. Ki values for compounds were calculated from observed IC50 values and the Kd value of the radioligand using the Cheng-Prusoff equation (Cheng Y, and Prusoff W H., Biochemical Pharmacology, 1973, 22, 23, 3099-108). The receptor subtype selectivity was calculated as the ratio of Ki(xcex21)/Ki(xcex22). With the exception of Compounds 7, 12, and 17 the compounds tested demonstrated a selectivity of greater than about 10. Thus, a preferred group of compounds are compounds of formula (I) other than Compounds 7, 12 and 17, which demonstrate a selectivity of at least 10 in Test A.
For the determination of agonist potencies, a HEK-293 cell line stably expressing cloned human xcex22 adrenergic receptor (clone H24.14) was grown to confluency in medium consisting of DMEM supplemented with 10% FBS and 500 xcexcg/mL Geneticin. The day before the assay, antibiotics were removed from the growth-medium.
cAMP assays were performed in a radioimmunoassay format using the Flashplate Adenylyl Cyclase Activation Assay System with 125I-cAMP (NEN SMP004, PerkinElmer Life Sciences Inc., Boston, Mass.), according to the manufacturers instructions.
On the day of the assay, cells were rinsed once with PBS, lifted with Versene 1:5,000 (0.2 g/L EDTA in PBS) and counted. Cells were pelleted by centrifugation at 1,000 rpm and resuspended in stimulation buffer prewarmed to 37xc2x0 C. at a final concentration of 800,000 cells/mL. Cells were used at a final concentration of 40,000 cells/well in the assay. Compounds were dissolved to a concentration of 10 mM in dissolving buffer (25 mM Gly-HCl pH 3.0 with 50% DMSO), then diluted to 1 mM in 50 mM Gly-HCl pH 3.0, and from there serially diluted into assay buffer (75 mM Tris/HCl pH 7.4 @ 25xc2x0 C., 12.5 mM MgCl2, 1 mM EDTA, 0.2% BSA). Compounds were tested in the assay at 10 different concentrations, ranging from 2.5 xcexcM to 9.5 pM. Reactions were incubated for 10 min at 37xc2x0 C. and stopped by addition of 100 xcexcl ice-cold detection buffer. Plates were sealed, incubated over night at 4xc2x0 C. and counted the next morning in a topcount scintillation counter (Packard BioScience Co., Meriden, Conn.). The amount of cAMP produced per mL of reaction was calculated based on the counts observed for the samples and cAMP standards, as described in the manufacturer""s user manual. Data were analyzed by nonlinear regression analysis with the GraphPad Prism Software package (GraphPad Software, Inc., San Diego, Calif.) using the 4-parameter model for sigmoidal dose-response with variable slope. Agonist potencies were expressed as pEC50 values. All of the compounds tested demonstrated pEC50 values greater than about 7. Preferred compounds of formula (I) demonstrate pEC50 values greater than about 8 in Test B. More preferred compounds of formula (I) are compounds that demonstrate pEC50 values greater than about 9.2 in Test B.
The following synthetic examples are offered to illustrate the invention, and are not to be construed in any way as limiting the scope of the invention.